Extensive multiplexing of protein nucleic-acid interactions to comprehensively study gene expression regulation from chromatin to mRNA degradation

项目摘要 通过涉及染色质的机制，基因的表达在RNA和蛋白质水平上都受到严格控制 修饰、转录调控、信使核糖核酸剪接、加工、翻译和降解。这其中的每一个 过程受核酸-蛋白质相互作用(DNA-蛋白质和RNA-蛋白质)的调节。因此，在那里 科学界做出了巨大努力，以全面描绘这些相互作用，包括 主要的国际研究努力(例如，ENCODE、路线图、表观基因组学)侧重于产生参考 特定单元格类型的映射。然而，由于这些结合图谱对单个细胞类型具有高度特异性， 迫切需要能够为任何感兴趣的细胞类型生成全面的基因组图谱- 包括原代细胞类型、疾病模型或其他稀有细胞群--在单个实验室内。这个目标 仍然具有挑战性，因为现有的分析一次只能绘制一种蛋白质的相互作用图，并且 因此，价格高得令人望而却步。 为了解决这些问题，该提案将基于我们的拆分池开发一种高度创新的技术 条形码策略(SPRITE)，使用高通量绘制多路蛋白质-核酸相互作用图 测序。拟议的Hi-P技术将用于建立：(I)高度多路复用的eCLIP-seq方法 将多达数百个RNA结合蛋白同时映射到它们的RNA结合部位，(Ii)高度多元化 芯片序列方法将多达数百种DNA结合蛋白和组蛋白修饰映射到它们的DNA结合 以及(Iii)在许多样本中绘制这些多个蛋白质-核酸相互作用图的方法 这些罕见的细胞类型，同时。 这项拟议的技术代表着一项重大进步--它将极大地扩大现有方法的规模 并创造出目前无法实现的新能力。这些工具将使个人研究人员能够 在特定的生物学和疾病背景下生成详细的基因组数据集，这些数据集在大小和 复杂性超过了ENCODE项目产生的复杂性，而成本只有它的一小部分。更广泛地说，我们预计 这些工具将导致对基因调控和人类疾病的关键新见解。

Project Summary Gene expression is tightly controlled at both the RNA and protein level by mechanisms involving chromatin modification, transcriptional regulation, mRNA splicing, processing, translation and degradation. Each of these processes are regulated by nucleic acid-protein interactions (DNA-protein and RNA-protein). Accordingly, there have been tremendous efforts in the scientific community to comprehensively map these interactions, including major international research efforts (e.g. ENCODE, RoadMap Epigenomics) focused on generating reference maps for specific cell types. However, because these binding maps are highly specific for individual cell types, there is a critical need to enable the generation of comprehensive genomic maps for any cell type of interest – including primary cell types, disease models, or other rare cell populations – within an individual lab. This goal remains challenging because existing assays can only map interactions of a single protein at a time and are therefore prohibitively expensive. To address these issues, this proposal will develop a highly innovative technology based on our split-pool barcoding strategy (SPRITE) that maps multiway protein-nucleic acid interactions using high throughput sequencing. The proposed Hi-P technology will be used to establish: (i) a highly multiplexed eCLIP-seq method to map up to hundreds of RNA binding proteins simultaneously to their RNA binding sites, (ii) a highly multiplexed ChIP-seq method to map up to hundreds of DNA binding proteins and histone modifications to their DNA binding sites, and (iii) methods to map these multiple protein-nucleic acid interactions across many samples, among these rare cell types, simultaneously. The proposed technology represents a major advance – it will dramatically increase the scale of existing methods and create new capabilities that are currently not possible. These tools will empower individual researchers to generate detailed genomic datasets in specific biological and disease contexts that are comparable in size and complexity to those generated by the ENCODE project at a tiny fraction of its cost. More generally, we anticipate that these tools will lead to critical new insights into gene regulation and human disease.